This invention relates to potassium channel subunit polypeptide and polynucleotide compositions, to the production of these compositions, and to the use of the compositions in the diagnosis, prevention, and treatment of disease.
Potassium channels are a heterogeneous group of ion channels that allow selective permeation of potassium ions across the plasma membrane, but differ in details of activation mechanism, voltage range of activity, and kinetic properties. (Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd Ed., Sinauer, Sunderland, Mass.; Latorre, R. and Miller, C. (1983) J. Memb. Biol. 7:11-30). They contribute to numerous physiological functions, for example, action potential repolarization, cardiac pacemaking, neuron bursting, muscle contraction, hormone secretion, vascular tone regulation, renal ion reabsorption, learning and memory, and cell growth and differentiation.
Voltage-gated potassium (Kv) channels are critical determinants of excitability in nerve and muscle cells, where they regulate impulse conduction, rhythmicity, and synaptic transmission. These channels form the largest and most diversified family of ion channels. At least six subfamilies of these channels have been identified: Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), Kv4 (Shal), KvLQT and EAG. Such channels are formed by the association of channel subunits, either of the same subunit (forming homomeric channels) or of different subunits (forming heteromeric channels). Kv channel subunits are characterized structurally by the presence of six transmembrane domains (S1-S6), one of which is highly positively charged, and a pore region between S5 and S6.
Regulation of Kv channel function can occur by association of different Kv subunits, resulting in heteromeric Kv channels having different conductance properties. Electrically silent Kv subunits, which by themselves do not form active homomeric channels, can modulate the conductance properties of Kv channels by associating with electrically active Kv subunits. For example, electrically-silent Kv subunits Kv6.1 and Kv8.1 modulate the conductance properties of Kv2 (Shab) channels by associating with Kv2.1 and Kv2.2 subunits, forming heteromeric Kv channels with conductance properties differing from the homomeric Kv2.1 and Kv2.2 channels (Post, M. A., et al. (1996) FEBS Lett. 399:177-182; Salinas. M. et al. (1997) J. Biol. Chem. 272:8774-8780)
Potassium channels are associated with a variety of disease states. In some diseases and disorders, abnormal ion channels are believed to be causative factors, while other diseases appear to arise from inappropriate regulation of otherwise normal ion channels. Diseases believed to have a particular association with potassium channels include neurological, cardiovascular, musculoskeletal, and proliferative disorders.
Potassium channel proteins are useful targets for drug therapy in a variety of neurological and vascular disease conditions. One such neurological condition is epilepsy, fundamentally a disease of neuronal overexcitability, resulting from excessive and synchronous firing of a large population of neurons in the cerebral cortex. In addition, many other neuropsychiatric diseases are consequences of abnormal neuronal activity in the cerebral cortex. Ideally, it would be desirable to treat epilepsy and related neuropsychiatric conditions with compounds that act selectively on ion channels in the cerebral cortex, to reduce side effects related to more generalized effects on the patient""s nervous system. To this end, it would be desirable to isolate, characterize, and recombinantly express human ion channel proteins (i) whose activities are related to neuronal excitability, and (ii) which are localized in the cerebral cortex.
A vascular condition associated with potassium ion current is pulmonary artery (PA) hypertension. Reduced voltage-dependent potassium current in PA smooth muscle cells leads to PA vasoconstriction, pulmonary hypertension and heart failure. It would therefore be desirable to treat pulmonary hypertension with compounds that restore potassium current in PA smooth muscle. Furthermore, it has been demonstrated that the appetite suppressant fenfluramine, which potentiates serotonin release in the brain, inhibits vascular smooth muscle potassium currents and is a causative agent in pulmonary hypertension (Weir E. K. et al. (1996) Circulation 94:2216-2220). It would therefore be desirable to isolate, characterize, and recombinantly express human ion channel proteins which are expressed in muscle tissues such as pulmonary artery smooth muscle, for use as a screening target to identify channel modulators useful in treating pulmonary hypertension and other cardiovascular and musculoskeletal disorders. Such channel proteins would also be useful in identifying compounds, such as serotonin release potentiators, which do not modulate such channels and thus lack hypertensive side-effects.
The invention includes proteins having sequence similarity to the Shab subfamily of voltage-gated potassium channel subunits and identified herein as Shab-like voltage-gated potassium channel subunits-1 and -2 (Kv-SL1 and Kv-SL2, and collectively as Kv-SL). The invention includes a substantially purified KV-SL protein having an amino acid sequence at least 85% identical to the sequence identified as SEQ ID NO:3 or SEQ ID NO:7 and which is capable of associating with one or more Kv subunits to form a Kv channel. In other embodiments, Kv-SL protein has a sequence at least 90% identical, preferably at least 95% identical to SEQ ID NO:3 or SEQ ID NO:7. In another embodiment, Kv-SL1 protein includes a portion between about amino acids 223 and 481 having a sequence selected from the group consisting of (a) the sequence between amino acids 223 and 481 of SEQ ID NO:3, (b) the sequence SEQ ID NO:5, and (c) internally consistent variations between sequences (a) and (b). In another embodiment, Kv-SL2 protein includes a portion between about amino acids 170 and 491 having a sequence selected from the group consisting of (a) the sequence between amino acids 170 and 491 of SEQ ID NO:7, (b) the sequence SEQ ID NO:9, and (c) internally consistent variations between sequences (a) and (b). In a more specific embodiment, Kv-SL1 protein has the sequence SEQ ID NO:3. In another specific embodiment, Kv-SL2 protein has the sequence SEQ ID NO:5 or SEQ ID NO:12. In another embodiment, Kv-SL protein is a human protein. The invention also includes fragments of Kv-SL protein, which are antigenic or which are capable of interacting with other proteins, peptides, or chemicals, such interaction which alters the functional properties or cellular/subcellular localization of Kv-SL protein or a Kv channel comprising Kv-SL. In one embodiment, the fragment corresponds to an intracellular domain of Kv-SL protein.
In another aspect the invention includes an isolated nucleic acid having a sequence which encodes Kv-SL as described above, or a sequence complementary to the Kv-SL coding sequence, and a composition comprising the nucleic acid. The nucleic acid may be mRNA, cRNA, DNA, cDNA, genomic DNA, or an antisense analog thereof. In various embodiments the nucleic acid may encode a Kv-SL protein having an amino acid sequence at least 85%, 90%, 95%, or 97% identical to SEQ ID NO:3 or SEQ ID NO:7. In another embodiment, the nucleic acid may encode a Kv-SL1 protein which includes a portion between about amino acids 223 and 481 having a sequence selected from the group consisting of (a) the sequence between amino acids 223 and 481 of SEQ ID NO:3, (b) the sequence SEQ ID NO:5, and (c) internally consistent variations between sequences (a) and (b). In another embodiment, the nucleic acid may encode a Kv-SL2 protein which includes a portion between about amino acids 170 and 491 having a sequence selected from the group consisting of (a) the sequence between amino acids 170 and 491 of SEQ ID NO:7, (b) the sequence SEQ ID NO:9, and (c) internally consistent variations between sequences (a) and (b). In more specific embodiments, the nucleic acid encodes a Kv-SL protein having the sequence SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:12. In another embodiment, the nucleic acid encodes a human Kv-SL protein. In other embodiments, the nucleic acid has the sequence identified as SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:11, or the complement thereof.
The invention also contemplates polynucleotides at least 12 nucleotides in length, preferably at least 15 nucleotides in length, more preferably at least 20, 25, 30, or 50 nucleotides in length, which hybridize under at least high-stringency conditions to any of the Kv-SL nucleic acids described above. The polynucleotide may be mRNA, cRNA, DNA, cDNA, genomic DNA, or an antisense analog thereof.
Also disclosed is a recombinant expression vector containing a nucleic acid encoding Kv-SL as described above, and, operably linked to the polynucleotide, regulatory elements effective for expression of the protein in a selected host. Preferred coding sequences are given above. In a related aspect, the invention includes a host cell, preferably a eukaryotic host cell, containing the vector.
The invention further includes a method for producing Kv-SL by recombinant techniques, by culturing recombinant host cells containing a nucleic acid encoding Kv-SL under conditions promoting expression of the protein, and subsequent recovery of the protein from the host cell.
In still another aspect, the invention includes an antibody specific for Kv-SL. The antibody has diagnostic and therapeutic applications, particularly in treating neurological and cardiovascular disorders. Treatment methods which employ antisense or coding sequence polynucleotides for inhibiting or enhancing levels of Kv-SL are also contemplated, as are treatment methods which employ antibodies specific for Kv-SL.
Diagnostic methods for detecting levels of Kv-SL in specific tissue samples, and for detecting levels of expression of Kv-SL in tissues, also form part of the invention. In one embodiment, a method of detecting a polynucleotide which encodes Kv-SL in a biological sample, involves the steps of: (a) hybridizing a polynucleotide, which is capable of hybridizing to a polynucleotide which encodes Kv-SL, to nucleic acid material of a biological sample, thereby forming a hybridization complex, and (b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of the polynucleotide encoding Kv-SL in the biological sample. Methods for detecting mutations in the coding region of Kv-SL are also contemplated.
Screening methods which employ Kv-SL for identifying a candidate compound which modulates the activity of Kv-SL also form part of the invention. An exemplary method includes (a) contacting a test compound with Kv-SL, under conditions in which an activity of Kv-SL can be measured, (b) measuring the effect of the test compound on the activity of Kv-SL, and (c) identifying the test compound as a candidate compound if its effect on the activity of Kv-SL is above a selected threshold level. The activity measured may be, for example, potassium conductance in a eukaryotic cell which expresses recombinant Kv-SL. In one embodiment, Kv-SL is a subunit of a heteromeric Kv channel. In another embodiment, the test compound is a component of a combinatorial library. In another embodiment, the test compound is an antibody specific for Kv-SL.
Screening methods which employ Kv-SL for identifying a candidate compound which does not modulate Kv-SL, for the purpose of identifying therapeutic compounds lacking Kv-SL-associated effects, also form part of the invention. An exemplary method includes (a) contacting a test compound with Kv-SL, under conditions in which an activity of Kv-SL can be measured, (b) measuring the effect of the test compound on the activity of Kv-SL, and (c) identifying the test compound as Kv-SL negative if its effect on the activity of Kv-SL is below a selected threshold level. The activity measured may be, for example, potassium conductance in a eukaryotic cell which expresses recombinant Kv-SL. In one embodiment, Kv-SL is a subunit of a heteromeric Kv channel. In another embodiment, the test compound is a component of a combinatorial library. In another embodiment, the test compound is a serotonin reuptake inhibitor or a serotonin release stimulator.
The invention also includes, in a related aspect, a compound identified by the screening methods described above, including a purified agonist (for example, a xe2x80x9cchannel openerxe2x80x9d) and a purified antagonist (for example, a xe2x80x9cchannel blockerxe2x80x9d). The invention further includes a purified antibody which specifically binds to a polypeptide described above.
The invention also includes methods to alter the expression level of Kv-SL by gene therapy techniques to achieve therapeutic benefit in patients.